Miocene sponge assemblages in the face of the Messinian Salinity Crisis—new data from the Atlanto-Mediterranean seaway

The Messinian Salinity Crisis is considered as one of the most influential Cenozoic events that impacted negatively on the benthic fauna of the Mediterranean area. Changing environmental conditions, including a sharp reduction of water exchange between the Mediterranean Sea and the Atlantic Ocean, altered the geographical ranges of many organisms, including sponges (Porifera). Here, we report a unique assemblage of isolated sponge spicules from the upper Miocene of southwestern Spain. The newly recognized sponge fauna was inhabiting the Guadalquivir Basin—the corridor between the Mediterranean and the Atlantic Ocean at that time. It represents a taxonomically rich sponge community that consisted of members of “soft” and “lithistid” demosponges and hexactinellids. Demosponges are represented by at least thirty-four taxa, while hexactinellids are significantly rarer; only six taxa have been identified. From among eighteen taxa recognized to the species level, at least eight seem to be inhabiting this area to these days; six are recorded from adjacent areas, such as the Western Mediterranean, South European Atlantic Shelf, and the Azores, and three are present in the Red Sea and/or the Northern Atlantic. Intriguingly, some taxa seem to have their closest relatives in distant areas, such as the Indo-Pacific and Japanese waters which suggests that the range of some once widely-distributed populations shrunk after the isolation of the Mediterranean and the Messinian Salinity Crisis, surviving to the present day only in refugia.


INTRODUCTION
Sponges (Porifera) are a diverse clade of sessile animals adapted to an aquatic lifestyle whose fossil record reaches at least the lowermost Cambrian (535 million years ago; Antcliffe, Callow & Brasier, 2014); as such, they are among the earliest-diverging multicellular organisms (Hamdi, Brasier & Jiang, 1989).Despite that sponges have often represented dominant components in marine environments, their record is substantially incomplete and the extinct communities are still poorly known.This is true especially for taxa that are being reported through their disassociated skeletal elements-the spicules,

Taxonomic assignment of loose sponge spicules
Throughout the Cenozoic, sponges maintained body plans and spicule morphologies that are broadly similar to, and often indistinguishable from, those of modern representatives of Porifera, prompting researchers to assign spicules obtained from numerous Cenozoic deposits to lineages (including species-level taxa) that are still present in modern aquatic environments (e.g., Costa et al., 2021;Łukowiak, 2015;Łukowiak et al., 2022b).Owing to the slow evolutionary rates of sponges and the conservative morphology of their skeletons, that can partially explain the longevity of some of their lineages (e.g., Jochum et al., 2012), we have included modern sponge taxa in our comparisons, focusing especially on those that are still present in the region.If no match was found, the studied spicules were compared with those that are present in taxa inhabiting distant geographical locations.

Geological background
The studied area is situated within the Guadalquivir Basin-the foreland basin of the Betic Cordillera located in South of Spain (Fig. 1).The Guadalquivir Basin is an important depression, elongated in NE-SW direction.The basinal sedimentary infill is limited to the south by the External Zone (or South Iberian Paleomargin) of the Betic Cordillera and to the north by the mountains of Sierra Morena, formed by Paleozoic and Mesozoic units of the Iberian Massif (Sanz de Galdeano & Vera, 1991& Vera, , 1992)).The Guadalquivir Basin formed as a foreland basin at the beginning of the late Miocene when the paleogeography of the Cordillera changed drastically once the westward drift of the Internal Zones ceased and the  Driussi et al., 2015 andOchoa et al., 2015).Towards the west the Guadalquivir basin and the position of the studied outcrops (MM: Martín Miguel, CV: Cerro Viejo).Figure modified from García-Veigas et al. (2020).
The dissociated sponge spicules originate from a Miocene unit known as moronitas or albarizas (a colloquial Spanish term referred to the white color of the strata) from the locality Jerez de la Frontera (Calderón & Paul, 1886).The typical facies of moronitas are laminated white (slightly yellow) marls, also called diatomaceous marls with a rich assemblage of calcareous (nanoplankton and Foraminifera) and siliceous (diatoms, radiolarians, silicoflagellates, and spicules of siliceous sponges) microfossils.From the biostratigraphical perspective, several authors assigned the moronitas to the Eocene, Oligocene, and Miocene (see the review in Colom & Gamundi, 1951), although the biostratigraphical data offered by Bustillo & López García (1995) establish three different episodes in the middle and late Miocene based on the diatom biozones.Diatoms and calcareous nannoplankton, as well as microfauna (radiolaria and foraminifera) are characteristic for a relatively shallow open marine basin (200-300 m) with a high organic production (IGME, 1988).The sedimentation with high diatomaceous content was the consequence of formation in a high-biogenic productivity area caused by coastal upwelling (Bustillo & García, 1997;López García, 1995).
The age and the paleoenvironment of the studied deposits (Martín Miguel and Cerro Viejo) was estimated based on the foraminiferids analysis.Each sample was wet-sieved to collect the >125 mm fraction, determining the presence of biostratigraphical marker species, the percentage of planktonic Foraminifera and the most representative benthic Foraminifera groups.The ratio of planktonic-to-benthic foraminiferids close to 30%, and the fact that benthic foraminiferids were dominated by cassidulinids, discorbids, buliminids, lagenids, and uvigerinids, suggests that the assemblages were inhabiting the outer shelf environment.The biostratigraphical analysis (the presence of Neogloboquadrina acostaensis) allowed establishing a Tortonian-Messinian (late Miocene) age for the studied sites.

Systematic paleontology
Class Demospongiae Sollas, 1885 Subclass Heteroscleromorpha Cárdenas, Pérez & Boury-Esnault, 2012Order Axinellida Lévi, 1953Family Raspailiidae Nardo, 1833 Genus Plocamione Topsent, 1927 Type species.Plocamione dirrhopalina Topsent, 1927 (type by monotypy).Plocamione cf.dirrhopalina Topsent, 1927 Figures 2A and 2B Material.Two spicules.Description.The spicules are stout curved acanthostyles.They are 120 and 150 µm long and possess ornamented heads on both spicule ends; in one case the heads are well-defined and the spicule is equipped with additional ornamented swelling in the spicule center.In the second case, only one head is well-developed and the other head smoothly passes into a median ornamented swelling which is less pronounced.Remarks.These spicules are of almost identical morphology as spicules of modern axinellid Plocamione, especially P. dirrhopalina (compare with Ridley & Duncan, 1881, pl. 23.19).This species is known today from the Mediterranean and the Azores (de Voogd et al., 2023) and is recorded from considerable depths of about 1,200 m (Topsent, 1928).

Figure 2C
Material.Single spicule.Description.Curved, 270 µm long style.The spicule's upper part is delicately ornamented with minute spines and strongly curved in a manner of a walking stick.The spicule thickness decreases gradually till the other end.Remarks.The studied curved style strongly resembles those of Rhabderemia fascicularis Topsent, 1927(Topsent, 1928, pl. X, fig. 25).However, the ornamentation of the modern species is of different character and does not cover the curved part of the spicule.This species is noted today from the N Atlantic (Azores, Canaries, and Madeira;de Voogd et al., 2023).There are also other species of Rhabderemia, e.g., R. typica or R. profunda which are characterized by presence of rhabdostyles in their skeletons but they usually are much smaller, or they are differently ornamented and curved.Likewise, the rhabdostyles may appear in some poecilosclerid and halichondrid species, but in this case assignment of the studied spicule to some unidentified species of Rhabderemia seem to be the most plausible.Remarks.Such spicule type characterizes sponges of the genus Bubaris.One species is noted from the same area (de Voogd et al., 2023) and possess identical anisoxeas of length 80 to 240 µm (Pulitzer-Finali, 1983, fig. 47).It is Bubaris subtyla and it is recorded from depth of 120-150 m (Pulitzer-Finali, 1983).There is also one bigger anisoxea that could have belonged to other species of Bubaris i.e., B. carcisis which is characterized by presence of longer spicules (600-1,870 µm in length; Vacelet, 1969, p. 36) and is noted from the Mediterranean and the Celtic Sea (de Voogd et al., 2023) but this assignment is tentative.Likewise, there are some other bubarid species that possess similar spicules in their skeletons, e.g., Auletta pedunculata (Topsent, 1896) or even some ancorinids (e.g., Jaspis) that are characterized by sinuous diactines and are recorded from the Mediterranean (de Voogd et al., 2023), so the unequivocal assignment of the longer diactines is not possible.Remarks.This strongly vermicular diactine characterizes sponges of the genus Monocrepidium.Among three species of Monocrepidium.One, M. vermiculatum is noted from the Mediterranean Sea (de Voogd et al., 2023).It also has been recorded in the N Atlantic and is restricted to rather deep waters (121-600 m depth; Alvarez & van Soest, 2002).

Figure 2D
Material.Single spicule.Description.Slender, spirally arranged microspined strongyle.This 65 µm long spicule is twisted with two twists and is delicately ornamented on the convex parts of the coils.Remarks.There are several species of clionaid genus Spiroxya noted from the Mediterranean and the Atlantic areas that are characterized by such spiral strongyles.For example, S. abyssorum (Carter, 1874) is noted from the N Atlantic (Celtic Sea;de Voogd et al., 2023), but their spiral strongyles are thinner and slenderer.On the other hand, Full-size  DOI: 10.7717/peerj.16277/fig- 2 S. heteroclita Topsent, 1896 which is noted from the Mediterranean, possess spicules of the same shape and length that the fossil one.Nevertheless, they seem not to be ornamented (compare with Topsent, 1900, fig. 11c).Spicules of Spiroxya corallophila (Calcinai, Cerrano & Bavestrello, 2002) are of similar shape, size and are sometimes microornamented (Calcinai, Cerrano & Bavestrello, 2002).This species is found in the western Mediterranean (de Voogd et al., 2023) and was retrieved from the coral living on the depth of 30-35 m (Calcinai, Cerrano & Bavestrello, 2002).On the other hand, Spiroxya levispira (Topsent, 1898) possess similarly ornament spiral strongyles as well (compare with van Soest & Beglinger, 2009, fig.3c) and it is noted from 80-700 m of the Mediterranean and N Atlantic Ocean (Rosell & Uriz, 2002;de Voogd et al., 2023).Spicules of shallow water Mediterranean species Spiroxya sarai (Melone, 1965) are also within the size range of the fossil spicule, but are smooth (Corriero, Abbiati & Santangelo, 1997).Likewise, a shallow water Spiroxya spiralis, which is noted today from the Azores and western Caribbean Sea, is characterized by the presence of microspined spiral strongyles of similar shape and size (Rützler et al., 2014).The studied strongyle belongs, most probably, to one of these species.
Family  72c).One of the spicules is characterized by the ends not in the same plane as the shaft (Fig. 2P).
Remarks.The size of diancistras suggests that they may belong to one of the two Mediterranean species characterized by big diancistras, namely Hamacantha (Vomerula) megancistra with 220-280-long diancistras (Pulitzer-Finali, 1978), or H. (Vomerula) papillata, with diancistras that can be 220-260 µm long (Boury-Esnault, Pansini & Uriz, 1994).Despite size similar to both these species, the presence of fimbriae on the spicule's shafts, suggests that they belong to the latter species.Hamacantha (Vomerula) papillata is recorded not only from the Mediterranean, but also from South European Atlantic Shelf from the depth range 185 to 1,600 m (de Voogd et al., 2023).
Remarks.These spicules may belong to Hamacantha (Hamacantha) johnsoni which is characterized by diancistras that are 113 to 167 µm long and are characterized by thin blade-like surfaces on the spicule ends (Boury-Esnault, Pansini & Uriz, 1994, fig. 73b).This species is recorded from the Mediterranean, including Alboran Sea and the South European Atlantic Shelf (Hajdu, 2002;de Voogd et al., 2023) Description.Heavily spined acanthoxea of a length of 250 mm.The coarse spines are chaotically arranged along the whole spicule length besides the pointed tips.They seem to get bigger to the spicule center and they are curved to the center as well.
Remarks.This kind of megascleres-acanthoxeas with smooth, pointed tips, is recorded in coelosphaerid genus Histodermella.The nearest to the study area species of Histodermella is Histodermella ingolfi, which is noted today from the N Atlantic Ocean (de Voogd et al., 2023).It possesses acanthoxeas with spine-free spicule tips and is of identical size (compare with Stephens, 1921, pl. 3, fig. 4).This species is also characterized by sigmas which can be up to 60 mm long.Histodermella ingolfi was reported living in considerable depths of about 700 to 1,400 m (Stephens, 1921).Description.Ornamented aciculospinorhabds, about 60 µm in length, with three subsidiary whorls and one apical whorl.In one spicule the subsidiary whorls divide dichotomously (Fig. 2V) and the apical whorl is a solitary, long spine (about 30 µm).
The other spicule is characterized by whorls which also divide but the apical whorl is shorter (20 µm) and divides at the middle point on three spines which are directed away from the shaft (Fig. 2W).
Remarks.There is a resemblance of these spicules to spicules of the sponges of the latrunculiid genus Cyclacanthia (Samaai, Govender & Kelly, 2004).Spicules of Cyclacanthia may also be ornamented and with apex with a single long spine and two subsidiary whorls (see e.g., C. bellae Samaai et al., 2003 1h) ich characterized by the same features, i.e., subsidiary and median whorls composed of slightly ornamented spines that divide; an apical whorl is made of single spine and a manubrium as a whorl of spines.On the other hand, the spicule resembles also isospinodiscorhabds of latrunculiid Cyclanthia bellae Samaai & Kelly (Samaai, Govender & Kelly, 2004, fig. 2f).Likewise, there are some Sceptrella species whose spicules show some resemblance to studied fossil aciculodiscorhabd.Also, some podospongiid spicules, e.g., of Red Sea inhabitant, Diacarnus ardoukobae Kelly-Borges & Vacelet, 1995 fall within the same morphological plan (compare with e.g., Kelly-Borges & Vacelet, 1995, fig. 3i).However, the assignment to latrunculiids is the most possible.Poecilosclerida indet.
Remarks.The tylotes are recorded from the poecilosclerid families Microcionidae, Coelosphaeridae, Tedanidae or Acarnidae.Also, acanthostyles are found in many poecilosclerid groups, e.g., Raspailiidae, Hymedesmiidae, Crellidae, and Microcionidae (Hooper & van Soest, 2002).Due to the general shape of these spicules, they assignment to the lower than poecilosclerid affinity is not possible.
Description.The spicules are stout, 90-µm-long strongyles with not numerous, big spines in two whorls arranged regularly along the spicule.The not well pronounced spicules' heads are also covered with spines.The spines are not numerous, about 15-20 µm long and are of the same size along the whole length of the spicule.
Remarks.These spicules resemble those of Podospongia loveni Barboza du Bocage, 1869 (compare with Kelly & Samaai, 2002, fig.1b)-a podospongiid sponge that inhabits today the Mediterranean Sea, including the Alboran Sea area (de Voogd et al., 2023).These strongyles also strongly resemble spicules of Sigmosceptrella quadrilobata Dendy, 1922 illustrated by Dendy (1922;pl. 18, fig. 4b).However, the same species illustrated by Kelly-Borges & Vacelet (1995) Hajdu, 2002) in contrast to fossil ones.However, the broken exotyle could have reach the size of the modern Mycale spicules when complete.This species is also characterized by presence of big, 250-400-mm-long anisochelae and about 200-mm-long sigmas (Stephens, 1921).Sigmas of comparable size had been found in the studied material as well (Fig. 2R) but dur to their very general shape their definite affinity cannot be established.M. (R.) marshallhalli is noted from the South European Atlantic Shelf and N Atlantic (de Voogd et al., 2023) from waters of about 75 to over 900 m of depth (Stephens, 1921).Description.An 85-µm-long and 18 µm-width placochela with delicate ornamentation along the inner part of the spicule alae.
The other spicule, the polytylote style with spined head, strongly resembles polytylote styles of A. tortillis (former A. polytylus; Pulitzer-Finali, 1983, fig.70).The delicate spination of the cladotylote head and the character of the rhabds is identical with these spicules.However, the size of the fossil spicule is a little bigger than of the modern Acarnus which is up to 430 µm.The definite assignment of this spicule is problematic, because similar spicules can be also found in few other Mediterranean species, e.g., Polymastia polytylota Vacelet, 1969, Latrunpagoda multirotalis (Topsent, 1927), and Hymedesmia (Stylopus) nigrescens (Topsent, 1925).However, none of these spicules possess spination on the spicule head.
Description.The 140 and 180-µm-long, slightly curved acanthostrongyles with spined surface.The spines are minute and placed without any order on the spicule surface, but on the faintly developed heads they are more densely arranged than on the rest of the spicule.On the spicule tips, the spines seem to be a bit curved and directed to the spicule center.
Remarks.These types of spicules are noted in the microcionid genus Clathria.The smooth stout style (Fig. 3E) shows great resemblance to spicules of Clathria (Clathria) hjorti (Arnesen, 1920)  Material.Single spicule.Description.Acanthoxea which is centrally bent and uniformly thinning to the spicule both ends.This 700-µm-long spicule possess minute spines regularly arranged along the whole length.
Remarks.This spicule resembles those of thoosid Alectona millari, however, the spicules of modern species are about two times smaller than the fossil one (Rützler, 2002b).This species is widely distributed all around the world (e.g., the Azores, Mediterranean Sea, including East European Atlantic Shelf; de Voogd et al., 2023); despite being mainly shallow-water species, several specimens of this sponge were found boring in deep-water corals around Madeira (Rützler, 2002b).Description.In the studied material there are several spicules that belong to suborder Astrophorina due to their tetraxial symmetry.Those are dichotriaenes (Figs.3W and 3X).
The first one is characterized by long, slender, dichotomously divided clads (that divide at 50 µm of length; the deuteroclads are at least 70 µm long but their exact length might be greater due to broken off tips).The other triaene is long-shafted orthomesotriaene which rhabd is 400 µm long and covered by small mammilliform outgrowths; the dichotomously divided clads are about 80 µm long (Fig. 3X).Another triaene recognized in the material is about 250 µm long, stout anatriaene with 50 µm long clads (Fig. 3Y).There are several calthrops in the studied material (e.g., Fig. 3V) which rays are 120 to 260 µm long and a single triod with about 220 µm long rays (Fig. 3U).
Remarks.The long triaenes (as well as anatriaenes assigned here to Tetillidae indet.)could have belonged to Geodia (family Geodiidae).However, lack of other spicule characteristic for this genus does not allow for the unquestionable assignment.Some of the described trienes could have belonged to tetillid sponges as well (e.g., Figs.3W and 3Y).
The assignment of triods and calthrop to a lower taxonomical level is not possible.
The first tetraclone's clads are about 100 µm long, smooth and strongly branching on their distal parts (Fig. 3AA).The other one is big, with one preserved clad of about 100 µm of length (the other two clads fragments are incomplete).The clads of this desma are rarely covered with wide and low projections and are branching on the tips (Fig. 3BB).The third desma is a rhizoclone, 220 × 170 µm, covered with numerous spines and outgrowths along the whole spicule (Fig. 3Z).
Remarks.The character of the studied spicules does not allow to assign them to any particular "lithistid" family, however, there is a high chance that the rhizoclone belonged to some sponge from the family Azoricidae or a rhizomorine "lithistid" (see Łukowiak et al., 2022a;Pisera & Lévi, 2002).

Figure 4A
Material.Single spicule.Description.A single fragment of lychniscosan skeleton which is characterized by a lantern-like structure that is created by fusion of lychniscosan hexactines.This dictyonal frame is about 100 µm in diameter and seem to come from the peripheral part of the framework as at least one of the "rays" seem to be originally preserved (unbroken) and measures about 100 µm of length.Remarks.This skeleton fragment can belong to one of the two lychniscosan families, Aulocystidae Sollas, 1887 or Diapleuridae Ijima, 1927.Both of them are characterized by presence of such joints within the skeleton.There are not many species within these two families and among them only two, Neoaulocystis polae (Ijima, 1927) and Neoaulocystis grayi (Bowerbank, 1869) (both aulocystids) are noted from the nearby areas.The first one is recorded from the Red Sea and the second one from the eastern Caribbean (de Voogd et al., 2023).Thus, it is highly possible that this dictional skeleton fragment belongs to one of these species.
Genus Description.There is a single hexactinellid microsclere noted in the studied material.This is discaster-a spicule about 110 µm in diameter, with spherically arranged, ornamented primary rays with blunt tips (Fig. 4S).There is also other spicule which is ornamented dermal stauractine with 160-µm-long rays that might belong to the same taxon (Fig. 4O).
Remarks.Microscleres which are quite unique spicules in fossil record give a high chance to be assigned to species level.In this case, the presence of very characteristic discaster together with stauractine allows to assig these spicules to a rossellid species Nodastrella nodastrella which is characterized by these two spicule types in its skeleton (compare with Dohrmann et al., 2012, fig. 1b and fig.2e).Moreover, there is a slender pentactine (Fig. 4Q) in the studied material that could have belonged to this species as well (compare with Topsent, 1915, fig. 2h).Likewise, it is possible that some of the diactines (Figs.4G-4I) found in the fossil material could also have belonged to N. nodastrella.This species is today recorded from N Atlantic (Azores, Canaries, and Madeira) and Eastern Coast of the Atlantic Ocean (i.e., Florida;de Voogd et al., 2023).

Figures 4L and 4T
Material.Two spicules.Description.The first spicule is ornamented pentactine with 80-µm-long, swollen ray tips (Fig. 4L).The second one is slender hypodermal pentactine with rays which are at least 250 µm long (all the rays are broken) (Fig. 4T).
Remarks.The ornamented pentactine can belong to some rossellid species of Crateromorpha or Acanthascus.The slender hypodermal pentactines appear in many species of rossellids (Tabachnick, 2002).However, it also appears in all three amphidicosid families Hyalonematidae, Monorhaphididae and Pheronematidae, so the assignment to rossellids is here tentative.
Order Sceptrulophora Mehl, 1992 Sceptrulophora indet.Figures 4M, 4N, 4P, 4R, 4U-4W.Material.Two spicules and four skeleton fragments.Description.First pentactine is characterized by actines located at one plane.They are about 170 µm long and delicately ornamented on the actins' tips.They also possess very characteristic, better pronounced ornamentation on the upper surface of the actines.The opposite actine is incomplete (Fig. 4M).The other pentactine possess about 150 µm long actines.This spicule is ornamented along the whole spicule length, but the ornamentation is better pronounced on the actines' tips.This spicule possesses a short knob on the apical part of the actine (not developed sixth actine; Fig. 4N).
There is also a single, fragmentarily preserved tyloscopule noted in the transmitted light (Fig. 4P).This very characteristic spicule is about 1,000 µm long and with well-developed, four ornamented rays with tylote tips.Among the three skeleton fragments, the first is characterized by delicately swollen nodes.
Remarks.Such ornamented pentactines appear in many families among Sceptrulophora, e. g., in Farreidae, Uncinateridae, or Euretidae.Sponges of the family Farreidae are quite abundant in the Mediterranean Sea (de Voogd et al., 2023), especially genus Farrea (Reiswig, 2002a).There are three species of Farrea living in the nearby area, e.g., in the Saharan Upwelling zone (F.occa Bowerbank, 1862) and in the Azores, Canaries, and Madeira (F.foliascens Topsent, 1906and F. laminaris Topsent, 1904(Topsent, 1928, pl. 4, fig. 6 and 8, respectively)).Thus, these spicules might have belonged to one of them but the assignment is not unequivocal.The pentactine with undeveloped sixth actine can be found in some sceptrulophoran families, e.g., Euretidae and Farreidae, but appear also in lyssacinosidan Rossellidae.The skeleton fragment with swollen nodes could belong to some of the euretid sponges, but its more precise assignment is not possible.
Tyloscopules appear in all sceptrulophorid families except Cribrospongiidae.These fragmentarily preserved frameworks are similar to frameworks of many sceptrulophoran groups, but some euretid (see Reiswig & Wheeler, 2002) and aphorocallistid (Reiswig, 2002b), sponges inhabit nearby areas, so it could have belonged to a sponge of one of these two families.The euretid Gymnorete alicei, which inhabits Azores and the Saharan Upwelling zone (de Voogd et al., 2023) possess very similar skeleton (Fig. 7b in Reiswig & Wheeler, 2002).Moreover, in the studied material we have found tyloscopule (Fig. 4P) which characterizes this species as well (Reiswig & Wheeler, 2002).Despite these similarities, the unequivocal assignment of the studied skeleton fragments is not possible.

Figures 4B and 4C
Material.Two skeleton fragments.Description.The fragmentary preserved skeletons; one of them is with a quadrangular mesh of about 60 × 20 µm in diameter and very characteristic four-fingered claw outgrowth (Fig. 4B).The other fragment is preserved as long fragment of a mesh and a five-rayed, delicately ornamented claw outgrowth (Fig. 4C).
Remarks.Despite these skeletons with characteristic outgrows are preserved only fragmentarily, they seem to be identical with skeleton fragments of modern uncinaterid species Tretopleura candelabrum (compare with Ijima, 1927, pl.XXII, fig.1).Today, this species which is the only known representative of this family, is recorded from the Banda Sea (de Voogd et al., 2023).

Figures 4D-4K
Material.Seven spicules.Description.There are three big, up to 1,000 µm long, diactines which apparently are reduced forms (by reduction of four actines) of hexactines.They are characterized by slightly curved axis and rounded actines tips.These spicules are sculptured with relief increasing on the actines' tips (Figs.4G-4I).
Remarks.The big diactines resemble the spicules of sponges of the family Aphrocallistidae, especially genus Aphrocallistes.Likewise, the pinnular hexactines found in the fossil material are of comparable shape (but bigger) of pinules of some modern Aphrocallistes, e. g., A. beatrix Gray, 1858.This species is noted from the studied area (de Voogd et al., 2023;compare with Lopes, Hajdu & Reiswig, 2005, fig.3).On the other hand, similar diactines can be found in other families e.g., Rossellidae, Leucopsacidae, Euplectellidae, or Hyalonematidae.The same is true for dermal pinules; they are recorded from families Euretidae, Pheronematidae, and Hyalonematidae (Reiswig & Wheeler, 2002;Tabachnick, 2002).

DISCUSSION
Our study shows that the Atlantic part of the Atlanto-Mediterranean Seaway was inhabited by unexpectedly rich and diverse sponge fauna about 11.6-5.3 million years ago (Tortonian-Messinian).It comprised members of two sponge classes, Hexactinellida and Demospongiae, and included at least three "lithistids".With respect to demosponges, we recognized at least thirty-four taxa.Of those, 17 could have been assigned to the species level and eight to the genus level.Hexactinellids were less common; still, they were represented by at least six taxa, including one that can be tentatively assigned to the species level and one that is assignable to the genus level.

Comparisons with other Miocene sponge faunas of Europe
The sponges described herein form the richest assemblage that has been reported from the Miocene of Europe to date.Several occurrences of dissociated sponge spicules have been reported from the European strata (for more details see Table S1).These include the sponge assemblage described by Pisera, Cachao & da Silva (2006) from Portugal that comprised "lithistids" (Rhizomorina), hadromerids (Alectona wallichii), spirophorids (Samus), astrophorids (Geodia and Erylus), and hexactinellids (Lychssacinosa).Of the taxa recognized in the material of Pisera, Cachao & da Silva (2006) only "lithistids" and hadromerids have been observed in our assemblage.In turn, the early Messinian sponge assemblage from the Turre Formation (Carboneras-Níjar basin, S Spain) described by Hoffmann et al. (2020), which was part of the northern edge of the Mediterranean basin at that time, includes three taxa.The demosponges from the Turre Formation are represented by triactinal tetraxons and calthrops, which were assigned by the authors to pachastrellids, whereas hexactinellids were represented by euretids and lychniscosans.While the pachastrellid spicules do not seem to have equivalents in our material, the hexactinellid skeleton fragment assigned by Hoffmann et al. (2020) to Euretidae shows resemblance to fragments found in our material.Lychniscosans was confirmed in both these assemblages as well.Also, the spicule assemblage from the Messinian of N Italy, despite recognizing 14 taxa (Costa et al., 2021), has not had much in common with our material.Only the spherasters of Tethya can be found in both these assemblages.This might be due to a shallower character of this Mediterranean assemblage.Surprisingly, in terms of shared taxa, the studied assemblage exhibits the greatest similarity with the middle Miocene sponge fauna of Paratethys (Vienna Basin, Slovakia) described by Łukowiak, Pisera & Schlögl (2014).This assemblage was interpreted to have originated from deep waters and contained at least thirteen spicule types.Among them, at least five seem to be common for these two assemblages (those belonging to Anulastrella, Thrombus, Alectona, Monocrepidium, and Lychniscosida, as well as anthasters similar to those of Tethya).
In terms of bathymetry, the studied assemblage shows a mixture of species typical for shallow waters (e.g., Mycale (Mycale) grandis, Sceptrella biannulata, Placospongia decorticans) and species characteristic for waters of considerable depths (e.g., Hamacantha (H.) lundbecki, Histodermella ingolfi, Discorhabdella tuberosocapitata, Thrombus abyssi).This discordance in bathymetrical preferences among the recognized taxa might be explained either by mixed character of the assemblage (e.g., due to transportation), different bathymetric preferences of some of the recognized taxa in the past, or specific water conditions in the studied area.We are inclined towards the third option because the coastal upwelling that took place in this part of the basin (López García, 1995) has likely changed water conditions that enabled the taxa with deep-water preferences to migrate into neritic depths during the late Miocene.
There is a general trend that indicates a depletion of the Mediterranean sponge fauna as we approach the MSC.Interestingly, the sponge fauna that inhabited the corridor between the Mediterranean and the Atlantic Ocean was doing well before as well as close to the MSC, being very diverse and rich.
Only four taxa seem to have no close relatives in adjacent waters and are possibly closer to sponges inhabiting distant parts of the world.In the case of spicules assigned to Acarnus sp., the morphologically closest morphotypes belong to Acarnus claudei-a species noted form S African coast (de Voogd et al., 2023).The oxyasters that we assigned to Tethya sp. also exhibit the greatest resemblance to spicules of distant, Indo-Pacific, species of Tethya (T.wilhelma and T. minuta).The same is true for hexactinellid skeletal fragments assigned to Tretopleura sp.The three known species belonging to this genus are currently recorded from the Banda Sea and W Pacific Ocean (de Voogd et al., 2023).Similarly, the spicule assigned here to Discorhabdella sp., despite showing some resemblance to the Mediterranean species, is most similar to those of D. hispida and D. misakiensis from Japan (Ise et al., 2021).This might indicate that the populations of species of Acarnus, Tretopleura, Tethya, and Discorhabdella had a wider geographic range in the past.This is the second case when spicules of the Alboran Sea species of Discorhabdella are more similar to geographically distant relatives than to species inhabiting neighboring areas (for more details see Boury-Esnault, Pansini & Uriz, 1992).
In turn, the contemporary geographical range of Mycale (Mycale) grandis covers the area of the Red Sea, East Africa coasts, Indonesian, and Pacific waters but not the E Atlantic (de Voogd et al., 2023).Its presence in East Atlantic, recognized during our study, extends the range of Mycale (Mycale) grandis to the majority of sea waters around the equator.Likewise, the modern distribution of Alectona millari, which is recorded from the N and E Atlantic Ocean through the Mediterranean, and up to Levantine Sea, must be also a Tethyan relic as this species (or spicules of this species) is recorded from the Miocene deposits of paleo-Mediterranean (Costa et al., 2021) and Eocene of Australia (Łukowiak, 2015).
The Tethyan origin of the modern distribution of some sponge taxa has already been postulated by many authors, e.g., Boury-Esnault, Pansini & Uriz (1992), and widely discussed by Łukowiak (2015), Łukowiak et al. (2022b).The studied assemblage provides new data to the discussion about the Tethyan relicts of the pre-Messinian biota.

CONCLUSIONS
We have recognized a rich and diverse sponge fauna from the Guadalquivir Basin, southwestern Spain, that has thrived before the onset of the Messinian Salinity Crisis.The community consisted of at least thirty-four taxa of demosponges and six hexactinellids.
From among eighteen taxa recognized to the species level, at least eight seem to be inhabiting this area to these days and many other of the studied sponges are recorded from adjacent areas, such as the Western Mediterranean, South European Atlantic Shelf, and the Azores.However, a group of sponge taxa identified in the studied sample were probably more closely connected to Indo-Pacific species, than to the species from the area.Such findings indicate that their geographical range has been wider in the geological past.The modern distribution of these taxa, which differs from their Miocene range, is an effect of a disruption after the closure of the Gibraltar strait.The shrinking ranges and disappearance from the Mediterranean Sea, resulting in disjunct modern distribution of some taxa, was likely the effect of the unfavorable conditions that took place in this area during the Messinian Salinity Crisis.
Gerardo Meiro performed the experiments, authored or reviewed drafts of the article, gathered and photographed material, and approved the final draft.Beltrán Peña performed the experiments, authored or reviewed drafts of the article, gathered material, and approved the final draft.Perfecto Villanueva Guimerans performed the experiments, analyzed the data, authored or reviewed drafts of the article, and approved the final draft.Hugo Corbí analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.

Field Study Permissions
The following information was supplied relating to field study approvals (i.e., approving body and any reference numbers): Verbal permit of Rafael Rendon Gomez-general Director of Bodegas Fundador
(de Voogd et al., 2023)om the Alboran Sea and N Atlantic Ocean so probably the spicules belong to this species(de Voogd et al., 2023).One tylostyle and one acantotylostyle.Description.A tylostyle fragment is 600 µm long, but the missing tip could have made the spicule at least 100 µm longer.The tylostyle head which is about 25 µm in diameter consist of up to 10 short, mammilliform projections (Fig.2S).The other spicule is strongly ornamented stout, 170 µm long, acanthotylostyle with well-developed spiny head (Fig.2T).But spicules of the geographically distant relatives from Japan, e.g., D. hispida or D. misakiensis, possess acanthostyles that resemble those from the fossil material (compare withIse et al., 2021, fig.4a and 6a, b, respectively).The spicules might belong or to this species, or to other, unknown so far from this area species of Discorhabdella.
(de Voogd et al., 2023) al., 2020,  fig.14a).However, none out of four species of this genus is recorded today from the area of N Atlantic or the Mediterranean Sea.These sponges inhabit South African waters(de Voogd et al., 2023).The studied spicules may belong to one of the Latrunculia species as well, e.g., L. (A.) biformis or L. apicalis (compare withSim-Smith et al., 2022, fig.62f and 64,  respectively).However, all of them inhabit the Southern Hemisphere today(de Voogd et al., 2023).The aciculospinorhabds characterize also sponges from the family Podospongiidae.
(de Voogd et al., 2023)dospongiids only two are present in this area, namely Podospongia loveni Barboza du Bocage, 1869 (noted from the Mediterranean) and Neopodospongia normani(Stephens, 1915)recorded from the N Atlantic(de Voogd et al., 2023).The studied spicules are similar to those of P. loveni (compare withCristobo et al., 2009, fig.5), however, in contrast to spicules of P. loveni, described here spicules are ornamented.In contrast, aciculospinorhabds of N. normani, these spicules possess apical whorl made of three spines, but also one additional central spine and only one collar of whorls instead of two (compare with Stephens, 1915, pl.V, fig.2b).Description.Small, 115-µm-long aciculodiscorhabd.The spicule possesses two whorls (median/subsidiary) that consist of delicately ornamented, diverging dichotomously spines.The apical whorl also consists of a single spine with a small whorl of minute spines in the middle of the spine length.Manubrium comprises five, delicately ornamented, spines.Remarks.This spicule is similar morphologically to latrunculiid and podospongiid spicules.The latrunculiid genus Tsitsikamma (illustrated e.g., in Li,Kelly & Tasdemir,  2021, fig.
Desbruyeres, Segonzac & Bright, 2006;n the vicinity: G. laplani, G. solorzanoi (both from South European Atlantic Shelf), G. fimbriata (Celtic Sea), and G. voljuta (Azores;de Voogd et al., 2023).However, none of the spicules that characterize these species possess serration on the spicule ridges.In the family Cladorhizidae the placochelae appear only in one species of the genus of Asbestopluma and within the genus Euchelipluma.The latter one contains five species only and only one is recorded from the nearby area of Cape Verde on the Atlantic Ocean(de Voogd et al., 2023).This is Euchelipluma pristinaTopsent, 1909and is characterized by having spicules identical in shape and size with the distinctive serration on the spicule ridges (compare withDesbruyeres, Segonzac & Bright, 2006; fig.5f).
(de Voogd et al., 2023)) can be assigned to one of Acarnus species.In the Mediterranean Sea there are only two species of Acanus recorded, A. levii, and A. tortilisTopsent, 1892  (former A. polytylus).This second one is also recorded from the South European Atlantic Shelf(de Voogd et al., 2023).However, in both these species, the cladotylote's distal end is not developed as small mammilliform projections, but as small hooks.Beside this, the spicules' shaft is covered by rare spines which is not observed in the fossil spicules.The other species noted from the nearby area are A. thieleiLévi, 1958, A. wolffgangi Keller,  1889(both from the Red Sea), and A. souriei(Lévi, 1952) (from the Azores; deVoogd et al., 2023).The first one possesses similar but smaller cladotylotes (up to 280 µm; van Soest,Hooper & Hiemstra, 1991).Interestingly, the studied spicules are almost identical with cladotylotes of modern Acarnus claudei van Soest,Hooper & Hiemstra, 1991, but this species occurs recently only in the S Africa(de Voogd et al., 2023).Also A. erithacus which is recorded from western coasts of N America possess cladotylotes with ornamented head(van Soest, Hooper & Hiemstra, (Bowerbank, 1866) 2023)r & de Voogd, 2013, fig.48b and Topsent, 1925, fig.15c).All these species are noted from the Mediterranean Sea and the last two were recorded also from the East European Atlantic Shelf(de Voogd et al., 2023).There are some other sponge species as well, that possess similar spicules, e.g., hymedesmiid Plocamionida ambigua(Bowerbank, 1866)or myxillid Ectyonopsis ramosa Carter, 1883, but spicules of these species are equipped with better developed spines (compare with van Soest, 2002, fig.2g).Still, the assignment of studied spicules to Antho seem the most adequate.However, due to lack of other spicule types that would allow the identification of species, the assignment only to genus level is possible.The other spicule noted in the fossil state, namely the smooth style (Fig.3E) shows some resemblance to some species of Plocamia and Antho which are also characterized by presence of acanthostrongyles (compare withvan Soest, Beglinger & de  Voogd, 2013, fig.46, 47, 48).
(Carter, 1880de Voogd et al., 2023)ic Ocean(de Voogd et al., 2023), C. (C.) coralloides, noted from the Adriatic Sea, and C. (C.) arecifera noted from the Azores (compare withvan Soest, Beglinger & de Voogd, 2013, fig.4a, 6 and 7c, respectively).The style with ornamented tip (Fig.3F) appear e.g., in Clathria (Microciona) capverdensis or Clathria (Microciona) boavistae; both are noted from Cape Verde (van Soest, Beglinger The other acanthostyles (fig.3H-J) could also have belonged to some Clathria or Antho species due to their shape and ornamented heads.It is worth noting that the character and general morphology of the studied styles does not allow to assign them to any species with certainty.Moreover, the Clathria species in general, are very numerous in the areas adjacent to the study area.There are several oxyasters with a diameter ranging from 60 to 130 µm, and with conical rays; some of the rays are diverging at the end.Remarks.Such characteristic oxyasters can be found in many species of this genus, e.g., in Tethya wilhelma and T. minuta.These two species possess spicules with divided/split ray Very characteristic, annulate triods with uneven, 150 to 190 µm long rays with well-defined narrow, smooth annuli.There are about 20 to 25 annuli on each ray, but not all of them continue on the whole ray diameter.The annuli cover the whole length of the rays including the point of contact of the rays.Remarks.The annulate triods resemble spicules of the theneid sponges Annulastrella ornata(Sollas, 1888)and A. annulata(Carter, 1880).The spicules of A. ornata are of comparable size, however they seem to be less annulated.Also A. verruculosa (Pulitzer- Finali, 1983), which is noted from the Mediterranean is similar in terms of shape, but smaller (rays up to 80 µm long).Today, Annulastrella ornata is known today from, among others, the western Mediterranean(Alboran Sea;de Voogd et al., 2023).
(de Voogd et al., 2023)ed, spiny triaenes with clads divided trichotomously called acanthotrichotriaenes.The clads are about 30 µm in length divide on the first 10 µm of their length.Remarks.The described acanthotrichotriaenes are of identical shape, ornamentation and size with those of the Recent species Thrombus abyssi Carter, 1873.Today, T. abyssi inhabits the Mediterranean and the Atlantic, including East European Atlantic Shelf(de Voogd et al., 2023).